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UNIVERSITI PUTRA MALAYSIA

STRUCTURAL AND BIOPHYSICAL CHARACTERISATIONS OF MeCP2 MBD MUTANTS THAT CORRELATE WITH RETT SYNDROME

CHIA JYH YEA

FPSK(M) 2016 47

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MBD MUTANTS THAT CORRELATE WITH RETT SYNDROME

By

CHIA JYH YEA

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Master of Science

January 2016

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in

fulfilment of the requirement for the Degree of Master of Science

STRUCTURAL AND BIOPHYSICAL CHARACTERISATIONS OF MeCP2

MBD MUTANTS THAT CORRELATE WITH RETT SYNDROME

By

CHIA JYH YEA

January 2016

Chairman : Ho Kok Lian, PhD

Faculty : Medicine and Health Sciences

Methyl-CpG binding protein (MBD) family consists of Methyl-CpG Binding Protein 2

(MeCP2), Methyl-CpG Binding Domain Protein 1 (MBD1), MBD2, MBD3 and MBD4

where MeCP2 is the prototype of the family. MeCP2 contains several domains: (a) a

methyl-CpG binding domain (MBD), (b) a transcriptional repression domain (TRD), (c)

two AT hooks and (d) a nuclear localisation signal (NLS). MeCP2 binds to methylated

DNA and represses the transcription of the associated genes. Mutations in MECP2 lead

to Rett syndrome (RTT), which is characterised by progressive neuro-developmental

disorder in early childhood of females. Previous studies revealed that most RTT

missense mutations alter the protein conformation and subsequently interferes the

methyl-CpG recognition. To understand how the structural changes contribute to RTT,

the 3-dimensional structure of these mutants need to be elucidated. Therefore, it is of interest to study the structure of RTT related MBD in complex with methylated DNA

using X-ray crystallography and characterised the DNA-protein binding with some

biophysical assays. Since more than 50% of the missense mutations occur within the

MBD domain. Out of the 8 hot RTT spots within this domain, RTT mutants D97E,

A140V, Y141C, P152R and D156E were included in this study. In band shift assay,

wild-type MBD complexed with DNA was significantly shifted compared to A140V

and D97E while other mutants were not significantly shifted. In SPR, wild-type MBD

showed the highest affinity towards the DNA followed by A140V (KD: 0.28 µM).

Circular dichrosim (CD) analysis revealed that the secondary structures of A140V,

Y141C, P152R and D156E are highly similar to wild-type MBD (14.7 % -helix, 25.2 %

-strand, and 60.1 % turns and unordered) except for D97E which showed 31 % -

helix, 6.5 % -strand, 62.5 % turns and unordered. The complex of MBD140V with methylated DNA was crystallised and diffracted X-ray to 2.2 Å resolution. The co-

crystal belongs to monoclinic space group C2, with unit cell parameters of a=78.66 Å,

b=53.49 Å, c=62.78 Å, ==90 ° and β=132.47 °. X-ray analysis revealed that the MBD domain was not altered by mutation of Ala-140 to Val (A140V). However,

additional water molecules were identified at the DNA-protein contact interface and around the DNA molecule. A narrow minor groove of A/T run was observed as a result

of additional bifurcated hydrogen bonds and vertical stacking of bases results from high

degree of propeller twist and heavy purine-purine stacking. Two hydration spines were

observed running down the wall of the minor groove. Each hydration spine is well

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arranged into two shells adopting a zig-zag arrangement. Hence, this finding provides

insights for the DNA geometry where the A/T run is geometrically stabilized by

extensive water network and is independent of the flanking nucleotide sequence, DNA

methylation and the bound MBD domain. The finding explores characteristics of the

methylated DNA containing A/T run, which provide the nucleotide sequence

preferences to MeCP2. In general, these additional molecular details could provide fundamental knowledge in RTT therapeutic approaches.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Master Sains

KAJIAN STRUKTUR DAN BIOFIZIKAL MUTAN MeCP2 MBD YANG

BERKAITAN DENGAN SINDROM RETT

Oleh

CHIA JYH YEA

Januari 2016

Pengerusi : Ho Kok Lian, PhD

Fakulti : Perubatan dan Sains Kesihatan

Keluarga protein pengikat metil-CpG (MBD) terdiri daripada protein Pengikat Metil-

CpG 2 (MeCP2), pengikat metil-CpG 1 (MBD1), MBD2, MBD3 dan MBD4 di mana

MeCP2 adalah prototaip keluarga. MeCP2 mengandungi beberapa domain: (a) domain pengikat metil-CpG (MBD), (b) domain penindasan transkripsi (TRD), (c) dua

pencangkuk AT dan (d) isyarat penempatan nuklear (NLS). Protein Pengikat Metil-

CpG 2 (MeCP2) mengikat DNA bermetil dan menindas transkripsi gen yang berkaitan.

Mutasi dalam MECP2 membawa kepada sindrom Rett (RTT), yang mempunyai ciri-

ciri progresif gangguan neuron dalam perkembangan awal kanak-kanak perempuan.

Kajian sebelum ini menunjukkan bahawa kebanyakan mutasi missense RTT mengubah

komformasi protein dan seterusnya mengganggu pengenalan metil-CpG. Untuk

memahami bagaimana perubahan struktur membawa kepada RTT, struktur 3-dimensi

mutan perlu dijelaskan. Oleh itu, adalah penting untuk mengkaji struktur RTT

berkaitan dengan MBD kompleks dengan DNA bermetil menggunakan sinaran-X

penghabluran dan mencirikan dengan beberapa analisis DNA biofizikal metilasi. Lebih

daripada 50% daripada mutasi missense berkelompok dalam domain MBD. Daripada 8 lokasi penting RTT dalam domain ini, mutan RTT termasuk D97E, A140V, Y141C,

P152R dan D156E telah dimasukkan dalam kajian ini. Dalam essei anjakan jalur,

Kompleks DNA-protein MBD jenis liar meranjak dengan ketara berbanding dengan

mutan A140V dan D97E manakala anjakan jalur mutan lain tidak ketara. Dalam SPR,

MBD jenis liar mempunyai daya afiniti ke arah DNA yang paling tinggi dan diikuti

oleh mutan A140V (KD: 0.28 µM). Analisis dikreisme membulat (CD) menunjukkan

bahawa struktur sekundur mutan RTT (A140V, Y141C, P152R dan D156E) adalah

menyerupai MBD jenis liar (14.7 % α-heliks, 25.2 % β-helai, dan 60.1 % tidak tersusun)

kecuali D97E yang menunjukkan 31 % α -heliks, 6.5 % β-helai, 62.5 % tidak tersusun.

Kompleks MBD140V dengan DNA bermetil telah dihablurkan dengan menggunakan 30 %

(w/v) PEG 2000 dan hablur telah dibelau oleh sinaran-X kepada resolusi 2.2 Å. Hablur ini tergolong dalam kumpulan ruang monoklinik C2, dengan parameter unit sel = 78.66

Å, b = 53.49 Å, c = 62.78 Å, α = γ = 90 ° dan β = 132.47 °. Analisis sinaran-X

mendedahkan bahawa domain MBD tidak berubah akibat mutasi Ala-140 kepada Val

(A140V). Walau bagaimanapun, lebih banyak molekul air telah dikenalpasti di antara

muka DNA-protein dan di sekitar molekul DNA. Lurah minor dengan urutan A/T yang

berulang telah dikenalpasti dengan tambahan ikatan hidrogen bercabang dan

penindihan nukleotida secara menegak yang berpunca daripada pusingan baling-baling

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yang berdarjah tinggi dan penindihan purina. Dua spina hidrasi didapati menuruni

dinding lurah minor. Setiap spina hidrasi tersusun dalam dua lapisan dengan

penyusunan zig-zag. Oleh itu, kajian ini dapat meningkatkan pemahaman dalam

geometri DNA di mana urutan A/T yang berulang adalah stabil akibat daripada

rangkaian molekul air dan bebas daripada mengapitnya urutan nukleotida, metilasi

DNA dan domain MBD yang terikat. Penemuan ini membongkarkan ciri-ciri DNA bermetil yang mengandungi urutan A/T berulang bagi membekalkan kegemaran

jujukan nukleotida kepada MeCP2. Secara umum, Informasi molekular yang didapati

dapat menawarkan perkembangan dalam permahaman asas dalam terapeutik RTT.

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ACKNOWLEDGEMENTS

With God‟s warmest blessing, there are some people who made my journey easier and

I would like to extend my greatest appreciation to those who helped me in completing

my journey. First and foremost, I would like to express my heartfelt gratitude to the

chairperson of the supervisory committee, Dr. Ho Kok Lian, for his endless support

and invaluable guidance throughout this study. He is always in an amiable manner with

his friendly gesture. All the gracious personalities of him have motivated me to become

a more down to earth and heart-warming person in the near future. In addition, his

impressive cognitive ability and intellectual maturity have also thought me the importance of critical and analytical reasoning, not only as a student point of view but

also as a researcher.

I would also like to extend my sincere appreciation to the members of the supervisory

committee, Prof. Dr. Tan Wen Siang and Assoc. Prof. Dr. Foo Hooi Ling, for their

wise opinions and constructive suggestions. Their endurance and boundless enthusiasm

in research are some of the characteristics that inspired me in the attempt to become a

diligent, persistent and successful researcher. I am also grateful to all my labmates,

especially Yoon Kam Yee; for their stimulating discussions, advice, their kind

hospitality, friendship and technical assistance. A million thanks goes to all of you who have brightened and amused me in one way or another for making my long exhausting

hours in lab a happy and pleasant one. Finally, my special thanks and deepest gratitude

are extended to my beloved family members for their understanding, perseverance,

support and endless love throughout my study.

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Ho Kok Lian, PhD

Senior Lecturer

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairperson)

Tan Wen Siang, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Member)

Foo Hooi Ling, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia (Member)

_____________________________

BUJANG KIM HUAT, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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Declaration by graduate student

I hereby confirm that:

this thesis is my original work

quotations, illustrations and citations have been duly referenced

the thesis has not been submitted previously or comcurrently for any other degree

at any institutions

intellectual property from the thesis and copyright of thesis are fully-owned by

Universiti Putra Malaysia, as according to the Universiti Putra Malaysia

(Research) Rules 2012;

written permission must be owned from supervisor and deputy vice –chancellor

(Research and innovation) before thesis is published (in the form of written,

printed or in electronic form) including books, journals, modules, proceedings,

popular writings, seminar papers, manuscripts, posters, reports, lecture notes,

learning modules or any other materials as stated in the Universiti Putra Malaysia

(Research) Rules 2012;

there is no plagiarism or data falsification/fabrication in the thesis, and scholarly

integrity is upheld as according to the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia

(Research) Rules 2012. The thesis has undergone plagiarism detection software

Signature: _______________________ Date: __________________

Name and Matric No.: Chia Jyh Yea , GS30076

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Declaration by Members of Supervisory Committee

This is to confirm that:

the research conducted and the writing of this thesis was under our supervision;

supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) are adhered to.

Signature:

Name of Chairman of Supervisory

Committee: Dr.Ho Kok Lian,

Signature:

Name of Member

of Supervisory

Committee: Professor

Dr.Tan Wen Siang

Signature:

Name of Member

of Supervisory

Committee:

Associate Professor

Dr.Foo Hooi Ling

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TABLE OF CONTENTS

Page

ABSTRACK i

ABSTRAK iii

ACKNOWLEDGEMENTS v

APPROVAL vi

DECLARATION viii

LIST OF TABLES xiiiLIST OF FIGURES xivLIST OF ABBREVIATIONS xviLIST OF AMINO ACIDS ABBREVIATIONS xix

CHAPTER

1 INTRODUCTION 1

1.1 General Introduction 1

1.2 Objectives 3

2 LITERATURE REVIEW 4

2.1 DNA methylation and epigenetics 4

2.2 DNA methylation represses gene expression 4

2.3 Methyl CpG Binding Protein Family 5

2.3.1 Identification of Methyl-CpG binding protein 5

2.3.2 MBD1 6

2.3.3 MBD2 and MBD3 6

2.3.4 MBD4 7

2.4 MeCP2 8

2.4.1 Methyl CpG binding protein 2 (MeCP2) 8

2.4.2 MeCP2 binding partners 8

2.4.3 MeCP2 and Rett syndrome 8

2.5 Structure of methyl-CpG binding domains 9

2.6 Protein chromatography 13

2.6.1 Immobilised Metal Affinity Chromatography

(IMAC) 13

2.6.2 Sephacryl S-200 gel filtration 13

2.7 Biophysical characterisations 14

2.7.1 Circular dichroism 14

2.7.2 Electrophoretic mobility shift assay (EMSA) 15

2.7.3 Surface Plasmon Resonance (SPR) 16

2.8 Protein Crystallography 16

2.8.1 Method of Crystallisation 17

2.8.2 Cryo-crystallography 20

2.8.3 X-ray diffraction 20

2.8.4 Data collection and processing strategy 22

2.8.5 Molecular replacement 23

2.8.6 Refinement and final validation 23

3 MATERIALS AND METHODS 25

3.1 Materials 25

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3.2 Construction of mutants 26

3.2.1 Plasmid extraction 26

3.2.2 Site-directed mutagenesis 26

3.2.3 Preparation of Competent cells 28

3.2.4 Transformation 28

3.3 Protein expression and purification 28

3.3.1 SDS-PAGE 29

3.4 Preparation of DNA double helix 30

3.5 Preparation of DNA-protein complex 30

3.6 Biophysical characterisations 30

3.6.1 Circular dichroism 30

3.6.2 Electrophoretic mobility shift assay (EMSA) 30

3.6.3 Surface plasmon resonance (SPR) 31

3.7 DNA-protein co-crystallisation 31

3.7.1 Crystallisation method, conditions and cryo-

protectant soaking 31

3.7.2 Data collection and processing 32

3.7.3 Molecular replacement, model building and

refinement 32

4 RESULTS AND DISCUSSION 33

4.1 Construction of MBD mutants 33

4.2 Expression of wild-type MBD and MBD mutants 36

4.3 Purification of wild-type MBD and MBD mutants 37

4.4 Characterisation of MBD mutants and in complex with

methylated DNA 39

4.4.1 Circular dichroism (CD) 39

4.4.2 Electrophoretic Mobility Shift assay (EMSA) 41

4.4.3 Surface Plasmon resonance (SPR) 42

4.5 Crystallisation 45

4.5.1 Crystallisation of MBD mutant D97E in complex

with methylated DNA 47

4.5.2 Crystallisation of MBD mutant A140V in

complex with methylated DNA 48

4.6 Data collection, processing and integration 49

4.6.1 Molecular replacement 52

4.6.2 Model building and refinement 52

4.7 Structural analysis of the MeCP2 MBD140V –methylated

DNA complex 56

4.7.1 Structural analysis of MBD bound methylated

DNA 58

4.7.2 Spine of hydration in the A/T run minor groove 60

4.8 DNA Geometry 65

4.8.1 Geometrical description of dinucleotides steps 65

4.8.2 Overall DNA geometry 66

4.8.3 Bend angle of DNA geometry 69

4.8.4 High degree of propeller twist 70

5 CONCLUSION AND FUTURE PERSPECTIVE 73

5.1 Conclusion 73

5.2 Future perspective 73

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REFERENCES 75

APPENDICES 85

BIODATA OF STUDENT 88

LIST OF PUBLICATIONS 89

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LIST OF TABLES

Table Page

3. 1 Common buffers and chemical compositions . 25

3. 2 Primers used to construct MBD mutants. 27

3. 3 Thermal profile used in QuikChange site-directed mutagenesis. 27

4. 1 Secondary structure analysis for wild-type MBD and MBD mutants. 40

4. 2 Data collection strategies. 49

4. 3 Crystallographic statistics for MBD in complex with methylated DNA. 50

4. 4 Thermal factor of TLS motion groups. 53

4. 5 Refinement statistics. 54

4. 6 Secondary structure of X-ray MBD140V–methylated DNA complex

structure analysed with PROMOTIF. 57

4. 7 MBD β-turns. 58

4. 8 Structured water molecules that bridge directly between protein and DNA

sugar-phosphate backbone. 60

4. 9 Water bridges at the AT run. 64

4. 10 Comparison of the minor and major groove width of the DNA fragment of

used in this study with at centres of dodecamer from Dickerson-Drew

(PDB:1BNA). 67

4. 11 Roll and Tilt of local base pair step parameter. 69

4. 12 Propeller twist of local base pair parameters. 71

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LIST OF FIGURES

Figure Page

2. 1 MeCP2 function in gene regulation and chromatin remodeling. 5

2. 2 The methyl-CpG binding protein family. 5

2. 3 Hydrolytic deamination. 7

2. 4 Eight hot mutation spots within the MBD domain. 9

2. 5 Overlay of MBD domains among MBD family members. 12

2. 6 Immobilised metal affinity chromatography. 13

2. 7 Size exclusion chromatography. 14

2. 8 Far UV CD spectra associated with various type of secondary structures. 15

2. 9 Schematic illustration of protein crystallisation phase diagram. 17

2. 10 Hanging drop vapour diffusion. 18

2. 11 Sitting drop vapour diffusion. 19

2. 12 Bragg's Law. 21

2. 13 Sphere of reflection or Ewald sphere construction. 22

4. 1 Side-directed mutagenesis of MBD. 35

4. 2 SDS-PAGE [15% (w/v)] analysis of expressed wild-type MBD. 36

4. 3 SDS-PAGE [15% (w/v)] of expressed wild-type MBD and MBD mutants.36

4. 4 SDS-PAGE [15% (w/v)] analysis of protein fractions separated from MBD

mutant A140V by IMAC affinity column. 37

4. 5 SDS-PAGE [15 % (w/v)] analysis of protein fraction separated from pooled

IMAC active fraction of MBD mutant A140V. 38

4. 6 Circular dichroism spectrum of wild-type MBD and MBD mutants. 40

4. 7 Band shift assay. 42

4. 8 Sensorgram of Surface plasmon resonance. 43

4. 9 Sensorgram of the binding of wild-type MBD and MBD mutants to Biotin-

methylated DNA. 44

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4. 10 Comparison of binding levels of the wild-type MBD and the MBD mutants to

Biotin-methylated DNA. 45

4. 11 Multiple sequence alignment of MBD domain of MBD proteins. 46

4. 12 Identity and similarity of MBD domain of MBD proteins. 47

4. 13 Crystallisation of D97E mutant in complex with methylated DNA. 48

4. 14 Crystallisation of A140V mutant in complex with methylated DNA. 49

4. 15 Data collection of crystal DNA-MBD140V. 51

4. 16 Model building of DNA-MBD140V. 52

4. 17 TLS motion groups of DNA-MBD140V model. 54

4. 18 Analysis of stereochemical properties of DNA-MBD140V X-ray structure. 55

4. 19 Overall X-ray structure of MBD140V in complex with methylated DNA

at 2.18 Å. 56

4. 20 E137 is stabilised by water molecules and amide groups. 59

4. 21 Hydrogen bonds between O2' of m5C8 with W63 and W64. 59

4. 22 Overall solvent structure of MBD140V in complex with methylated DNA. 62

4. 23 Bridging system in AT run. 63

4. 24 Geometrical description of DNA parameters. 65

4. 25 Graphical output of a Curves+ analysis for DNA. 66

4. 26 Overlay of idealised B-DNA (Blue) and X-ray DNA (Cyan) with A/T run

highlighted in red. 68

4. 27 Superimposed of A/T run of MBD140V with HMGA-1. 68

4. 28 Stereo view of the superposition of the A/T run. 70

4. 29 High degree of propeller twist at AT run base pair. 72

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LIST OF ABBREVIATIONS

α Alpha

Å Ångström

β Beta

µg microgram (10-6 g)

µL microliter (10-6 L)

µM micromolar (10-6 M)

BDNF Brain-derived neurotrophic factor

bp Basepair

BSA bovine serum albumin

ccc covalently closed circular

CCP4 Collaborative Computational Project Number 4

C-terminal carboxyl terminal

DNA Deoxy-ribonucleic acid

dNTP deoxynucleoside triphosphate

dsDNA double stranded DNA

DTT 1,4-dithiothreitol

EDTA ethylene diamine tetraacetic acid

EMSA Electrophoretic mobility shift assay

FPLC fast protein liquid chromatography

HDAC Histone deacetylase

HPLC high performance liquid chromatography

IMAC immobilised metal ion affinity chromatography

IPTG isopropyl-β-d-thiogalactopyranoside

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K Kelvin

Kb kilobase

Kd dissociation constant

kDA kilo Dalton

LB Luria broth

m5C 5’methyl cytosine

MBD Methyl-CpG Binding Domain

MBD1 Methyl-CpG Binding Domain Protein 1

MBD2 Methyl-CpG Binding Domain Protein 2

MBD3 Methyl-CpG Binding Domain Protein 3

MBD4 Methyl-CpG Binding Domain Protein 4

MeCP2 Methyl-CpG Binding Protein 2

MBP Methyl-CpG Binding Protein

Mg milligram (10-3 g)

Min minute

NDB Nucleic Acid Database

NLS Nuclear localisation Signal

nM nanomolar (10-9 M)

NTA Nitrilotriacetic acid

OD optical density

PAGE polyacrylamide gel electrophoresis

PCM1 Protein containing MBD 1

PCR Polymerase chain reaction

PDB Protein Database Bank

PEG Polyethylene glycol

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pI Isoelectric point

RMSD root mean square deviation

rpm revolutions per minute

RTT Rett Syndrome

s Second

SDS

SEC

sodium dodecyl sulphate

size exclusion chromatography

TBE Tris-buffered EDTA solution

TEMED Tetramethyl ethylenediamine

TRD Transcriptional repression domain

U Unit

UV Ultraviolet

v Volt

Vm Matthew’s coefficient

v/v volume/volume

w/v weight/volume

x g centrifugal force

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LIST OF AMINO ACIDS ABBREVIATIONS

One letter code Three letter code

Alanine A Ala

Arginine R Arg

Asparagine N Asn

Aspartic acid D Asp

Cysteine C Cys

Glutamic acid E Glu

Glutamine Q Gln

Glycine G Gly

Histidine H His

Isoleucine I Ile

Leucine L Leu

Lysine K Lys

Methionine M Met

Phenylalanine F Phe

Proline P Pro

Serine S Ser

Threonine T Thr

Tryptophan W Trp

Tyrosine Y Tyr

Valine V Val

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CHAPTER 1

INTRODUCTION

1.1 General Introduction

DNA methylation is an epigenetic signal that affects gene regulation, genomic stability

and chromatin structure in mammalian cells (Bird, 2002; Du et al., 2015). In most cases,

this signal can be read by a family of proteins that contains a common methyl-CpG

binding domain (MBD) (Hendrich and Bird, 1998). To date, five family members,

namely MeCP2, MBD1, MBD2, MBD3 and MBD4 have been identified, in which,

MeCP2 is the prototype of this family (Hendrich and Bird, 1998). MeCP2, MBD1 and

MBD2 are able to recruit co-repressor complexes that can inhibit transcription in concert

with chromatin modifiers (Scarsdale et al., 2011). Mammalian MBD3 does not bind to

methylated DNA in vitro and in vivo due to replacement of amino acid (K43H and Y47F)

which is critical for DNA binding (Fraga et al., 2003). MBD4 contains a thymine DNA-

glycosylase at the C-terminal region that can repair G-T mismatches via hydrolytic

deamination [(Refer Section 2.3.4 and Figure 2.3) (Neddermann et al., 1996)].

MeCP2 is a transcriptional repressor that contains several domains: (a) a methyl-CpG

binding domain (MBD), (b) a transcriptional repression domain (TRD), (c) AT hooks

and (d) a nuclear localisation signal (NLS) (Lewis et al., 1992; Nan and Bird, 2001; Xu

and Pozzo-Miller, 2013). MBD domain of MeCP2 is able to recognise methyl-CpG

containing DNA. TRD domain involves in recruitment of transcriptional co-repressors

such as mSin3A and histone deacetylases (HDACs) (Bienvenu et al., 2000). Two

putative NLSs facilitate nuclear localisation, which targets the protein into the cell

nucleus (Weaving et al., 2004) and the AT hooks are believed to interact with AT rich

region of the DNA (Klose et al., 2005). In addition, the AT hooks of MeCP2 bearing

amino acid sequences 185GRGRGRP191 and 265PKKRGRKP272 (superscript indicates

amino acid number) which are highly similar to the high mobility group with the AT

hook I chromosomal protein (HMCG-I) that is capable to bind to the minor groove of

the AT stretches (A/T run) of DNA and functionally (Aravind and Landsman, 1998;

Lewis et al., 1992; Nan et al., 1993; Reeves and Nissen, 1990). Baker et al. (2013)

demonstrated that the disruption of second conserved AT hook at the C-terminal region

of MeCP2 by truncation at R270X of MeCP2 led to failure in chromatin compaction and

localization of pericentric heterochromatin domain of -thalassemia mental retardation

syndrome X-linked (ATRX); a chromatin remodelling protein, with MeCP2, and caused

the R270X mice to exhibit Rett syndrome (RTT) phenotypes which is similar to MeCP2

knock-out mice (Baker et al., 2013; Xu and Pozzo-Miller, 2013).

The MBD domain of MeCP2 is able to recognise single methyl-CpG dinucleotide (Lewis

et al., 1992). The MBD domain alone is ample for the methylated DNA binding and

mutations in the MBD domain intercept its binding to methylated sequence (Baubec et

al., 2013; Yusufzai and Wolffe, 2000). MeCP2 mutation causes Rett syndrome (RTT); a

progressive neurodevelopmental disorder in early childhood, which leads to mental

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retardation in females, with a prevalence of 1 in 10,000-15,000 female births (Hagberg,

1985). RTT is caused by an X-linked mutation dominant inheritance with normally

lethality in males due to severe encephalopathy (Bianciardi et al., 2015; Bienvenu et al.,

2000; Zhao et al., 2015). Studies revealed that most RTT related missense mutations

alter the structure of the MBD domain and subsequently interrupt DNA recognition

properties (Kriaucionis and Bird, 2003; Kucukkal et al., 2015). Klose et al. (2005)

showed that an A/T run adjacent to the methyl-CpG is required to enhance the MeCP2

binding (Klose et al., 2005). Identified endogenous MeCP2 targeting genes such as brain

derived neurotropic factor (BDNF) promoter region contains high occurrences of A/T

runs closed to the methyl-CpGs (Chen et al., 2003; Martinowich et al., 2003). The A/T

run in the methylated DNA facilitated the co-crystallisation of MeCP2 MBD domain in

complex with methylated DNA used in this study. Due to the presence of AT hooks in

MeCP2 and the requirement of A/T run for maximal binding, it has been speculated that

the A/T run could interact with the AT hooks of the MeCP2. However, the characteristics

of A/T run which provide specificity for the MeCP2 to recognise the methyl groups

remained unclear. Therefore, it is of interest to elucidate the 3-dimensional structure of

RTT mutants in order to understand how the structural changes contribute to RTT and

the A/T run characteristics with MBD domain bound to its adjacent methyl-CpG

dinucleotide.

X-ray analysis of previous report on a MeCP2 MBD-methylated DNA complex revealed

that only a few residues are involved in direct contact with the DNA bases (Ho et al.,

2008). The methyl groups are recognised by the Arginine fingers of R111 and R133

while D121 is critical in maintaining the unique hydration pattern at the DNA-protein

interface. The unique water molecules distribution pattern is crucial to mediate methyl

group recognition (Ho et al., 2008). RTT mutations within the MBD domain of MeCP2,

however, are believed to alter the 3-dimensional structure of the protein and subsequently

affects DNA binding. Several critical mutations such as R111G, R133C, T158M and

D121G, which close to the DNA-protein contact region have been investigated (Free et

al., 2001; Meehan et al., 1992; Nan et al., 1993; Yusufzai and Wolffe, 2000). In this

study, other RTT mutations which are located distance from the DNA-protein contact

region have been studied. According to Wakefield and colleagues (1999), missense

mutations found in RTT usually do not specifically interrupt DNA recognition but may

result in structural changes in the domain (Bianciardi et al., 2015; Wakefield et al., 1999).

In order to further investigate the details on the structural changes and molecular

functional role, several mutants (D97E, A140V, Y141C, P152R and D156E) were

constructed and the DNA-MBD interactions were characterised with various biophysical

assays. A co-crystal structure was also elucidated, in which, more molecular details about

the DNA-protein complex have been revealed compared with previous reported structure.

In addition, the atomic details of the DNA geometry of the MBD bound A/T run are

highlighted in comparison with the A/T run of the free DNA double helices. In general,

these additional molecular details could provide fundamental knowledge in RTT

therapeutic approaches.

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1.2 Objectives

The general objective of this study was to explore and understand the atomic details

of Rett mutants in complex with methylated DNA. The specific objectives were:

1. To construct MBD mutants

2. To characterise the interactions of MBD mutants and methylated DNA

3. To crystallise the MBD mutants in complex with methylated DNA

4. To solve the X-ray structure of MBD mutants in complex with methylated DNA

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